Ab52623

Mice xenograft anti-PD-1 therapy study, western blot, circRNA immunoprecipitation (circRIP), RNA pull-down, and dual luciferase reporter assays were evaluated with SPSS software (19.0, SPSS, Inc., Chicago, IL) as described in our previously studies^{[8 (link),10 (link),14 (link)]}. Experimentation on C57BL/6 mice were approved by the Animal Experimentation Ethics Committee of Zhongshan Hospital, Fudan University. And the antibody used in western blot and circRIP assays were listed as follows: Tubulin (Abcam, ab52623), AGO2 (Abcam, ab186733), and SOX4 (Abcam, ab70598).

Cells were fixed with 4% paraformaldehyde and 0.1% Triton X‐100 in phosphate‐buffered saline (PBS) buffer for 20 min. Fixed cells were incubated for 1 h at room temperature in a blocking solution (PBS containing 0.5% FBS) to reduce nonspecific binding. Cells were treated with diluted primary antibody in the blocking solution overnight at 4°C, and with diluted secondary antibody in the blocking solution for 1 h at room temperature. The monoclonal antibody β‐Tubullin III (ab52623, RRID: AB 869991, 1:500; Abcam, Cambridge, MA, USA) was used as the primary antibody and Alexa Fluor‐conjugated IgG (ab150077, RRID: AB 2630356, 1:1,000; Abcam) as the secondary antibody. 4′,6‐diamidino‐2‐phenylindole was used to stain cell nuclei (blue) for 30 min at a concentration of 1.43 μM, and cells were imaged with a fluorescent microscope (Nikon).

Primary antibodies: Flag antibodies (M2, Sigma and MDBio, Taiwan), Actin monoclonal antibody (Millipore, Billerica, MA, USA), GATA1 antibody (MDBio), CLDN11 antibodies (Western/ab53041, Abcam, Cambridge, UK; IHC/SC25711, Santa Cruz, Dallas, TX), Tubulin alpha IB (ab108629, Abcam) and Tubulin beta III antibodies (ab52623, Abcam). Secondary antibodies: HRP-conjugated goat anti-rabbit and goat anti-mouse antibodies (Gene Teks) were used. Western blot results were visualized by ECL detection kit (Immobilon, Merck/Millipore, Germany). Recombinant proteins GST-GATA1 (H00002623, Abnova, Taiwan) and GST-CLDN11 (H00005010, Abnova) were used in EMSA and tubulin polymerization assay, respectively.

After treatment for 72 h, the mice were sacrificed, and the eyeballs (n = 3) were enucleated and subsequently fixed in 4% immunohistochemical fixation solution for 2 h. After rinsing with phosphate buffer solution (PBS) three times, all of the corneas were clipped along the margin of the keratosclera and then incubated with anti-β3-tubulin (1:500, ab52623, Abcam, Cambridge, United Kingdom), anti-SP (1:400, ab10353, Abcam) and anti-CGRP (1:400, 14959s, Cell Signaling Technology, United Kingdom) for 48 h at room temperature. After washing with PBS, the cornea was incubated with Alexa Fluor® 488 goat anti-guinea pig IgG H&L (1:500, ab10353, Abcam) and Alexa Fluor® 594 goat anti-rabbit IgG H&L (1:400, ZF-0516, ZSGB-BIO, China) at 4 °C overnight. After counterstaining with 4,6-diamidino-2-phenylindole (DAPI, ZSGB-BIO, China) to stain the nuclei, all of the images were captured by using a fluorescence microscope (Nikon, Tokyo, Japan). To compare the changes in corneal innervation, Image J and Neuron J software were used to calculate the nerve length in the positive area for β3-tubulin staining per sample. In the experiment, one representative image in the center of the cornea and four replicates captured in the peripheral area of cornea from three independent experiments per group were used for analysis.

The mouse xenograft tumors were fixed in paraformaldehyde for 1 h, followed by conventional paraffin‐embedding and sectioning (4 μm), and drying at 60℃. Paraffin sections were dewaxed with xylene and graded ethanol, and rinsed with distilled water. The slides were boiled in sodium citrate buffer (pH = 6.0, Solarbio, Beijing, China) for 10 min for antigen recovery. The sections were incubated in 3% hydrogen peroxide (Solarbio) for 10 min to block endogenous peroxidase activity and in 5% normal goat serum (ZGB‐BIO, Beijing, China) to block nonspecific protein binding sites. The sections were then incubated with antibodies against cleaved‐caspase‐3 (1:500; NB100‐56113, Novus Biologicals) and β‐Tubulin‐III (ab52623, RRID: AB_2819160, 1:800; Abcam) overnight at 4°C and subsequently incubated with goat antirabbit biotin‐conjugated secondary antibody (ab205718, 1:800; Abcam) for 0.5 h at room temperature. The reaction products were characterized using diaminobenzidine (Sigma‐Aldrich Chemical Company, St Louis, MO, USA). Nuclei were counted lightly with hematoxylin (Solarbio), and immunostaining was observed under a light microscopy (Olympus Optical Co., Ltd., Tokyo, Japan). The mean OD values of different sections were compared by Image‐pro Plus software.